【作者】 张倜；

【导师】 汤钊猷；

【作者基本信息】 复旦大学 ， 外科学， 2005， 博士

【摘要】 原发性肝癌是常见的恶性肿瘤,手术切除仍是最有效的治疗方法,但根治术后5年转移复发率高达60-70%,是影响肝癌远期疗效的主要原因。抑制肿瘤血管生成,可减少肿瘤赖以生存的营养供应,从而抑制肿瘤生长。对不能切除的肝癌和肝癌术后转移复发的预防,抗血管生成是一条有希望的途径,但由于缺乏肝癌血管特异的靶点,因而疗效有限。血管异质性是近年肿瘤血管生成研究的热点,从分子水平研究肿瘤血管的异质性,有助于监测肿瘤的演进,估计肿瘤的恶性程度,并可能为肿瘤治疗提供新靶点。迄今,已有结肠癌、肺癌及肾癌等肿瘤血管内皮细胞分子水平异质性研究的报道,尚未见肝癌相关的研究报道。本课题拟通过分离不同转移潜能裸鼠人肝癌的血管内皮细胞,检测其血管生成相关基因表达的改变,以期发现肝癌转移复发过程中,与血管生成密切相关的某些基因表达改变,为肝癌转移复发研究提供新的分子标记和治疗靶点,并进一步研究我们已证实的干扰素抑制肝癌转移复发与血管内皮的关系。 1.肿瘤血管内皮研究的平台建设一裸鼠人肝癌血管内皮细胞的分离与鉴定 血管内皮细胞的数量极为有限,其分离与纯化受多种因素影响,如何从肿瘤组织的多种混杂细胞中分离纯化内皮细胞成为问题的关键。我们采用CD31抗体交联免疫磁珠分选法,并加以优化,对裸小鼠原位人肝癌移植瘤进行血管内皮细胞分选;从肝癌组织中分离的细胞呈现典型的内皮细胞形态特征,免疫组化证实内皮细胞标记分子CD31及VE-Cadherin表达阳性,超过95%的阳性分选细胞呈乙酰化低密度脂蛋白(Dil-Ac-LDL)摄取实验阳性,RT-PCR示获得的细胞中内皮细胞标记分子CD31、VE-Cadherin及CD146的mRNA含量明显高于分离前,而肝癌细胞标记分子AFP及白细胞标记分子CD45在所分选的细胞中呈阴性表达。上述结果表明,采用优化的免疫磁分选法有效地分离纯化了肝癌组织中的血管内皮细胞,分离的内皮细胞可用于肝癌血管生成的相关研究。 2.不同转移潜能人肝癌相关血管内皮基因表达差异的研究 我所前期已建成具有不同转移潜能的人肝癌模型体系。我们对不同转移潜能肝癌来源的血管内皮细胞进行了基因表达分析。采用血管生成相关基因分类芯片分析时发现,不同转移潜能肝癌来源的血管内皮细胞存在差异基因表达。在96个候选基因中,有7个基因的表达与转移潜能变化一致,2个呈负相关(Adamts8,Serpinb5),5个呈正相关(Ctgf,Ets1,PDGFRα,TGFβ3,TIMP-2)。其中,PDGFRα更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Asia and Africa. Despite endeavors have been made to improve its prognosis, the overall survival, however, is still unsatisfied. The high rate of metastasis and recurrence after curative resection was responsible for the poor prognosis of resectable HCC.Anti-angiogenesis is an attractive treatment for HCC, because HCC is a typical hypervascular cancer, its growth, metastasis or even hepatocarcinogenesis depend on angiogenesis. However, the success of anti-angiogenesis treatment in HCC has been sparsely reported. The heterogeneity of tumor vessels, which has been widely accepted, may result in the un-universal response to anti-angiogenesis treatment. As a major part of tumor vessels, endothelial cells are heterogeneous in different organs, tissues and tumors. The distinct molecules on tumor derived endothelial cell (TEC) can either serve as diagnostic markers or therapeutic targets. Unfortunately, little information is available about the heterogeneity of the vascular endothelium in HCC. The aim of this study was to identify the metastasis-related genes in tumor endothelial cells from HCC.I. Isolation and verification of TEC from human HCC xenografts in nude miceAs a main target of anti-angiogenesis therapy, TECs represent only a minor fraction of the total cell population within tumor tissues. Along with the fact that endothelial cells are enmeshed in a complex tissue consisting of a variety of cells, analysis of TEC has long been considered a tough task due to the difficulty of isolation and purification. Here modified immunomagnetic methods using magnetic beads conjugated with anti-CD31 antibody were used to isolate vascular endothelial cells from HCC xenografts; the isolated endothelial cells presented typical "cobblestone" appearance. Internalization of acetylated low-density lipoprotein was positive in more than 95% of isolated cells. The endothelial cells were confirmed by immunofluorescence staining using endothelium specific antibody, CD31 and VE-Cadherin. RT-PCR analysis showed robust amplification of endothelial-specific transcripts, CD31, VE-Cadherinand CD 146 in isolated endothelial cells. Contamination of tumor cells or leucocytes was excluded by the negative expression of AFP or CD45, respectively. Thus, TEC can be isolated from HCC nodules by using modified immunomagnetic methods, and these cells can be used for angiogenesis related researches in HCC.II. Gene expression profiling of TEC from human HCC xenografts with different metastatic potentialA human HCC metastasis model system has been established at the author’s institution. TECs from HCC with different metastatic potential were used to analyze the angiogenesis related gene expression profile. cDNA array analysis revealed seven differentially expressed genes within 96 genes detected consistently presented in endothelial cells derived from HCC with different metastatic potential, in which Adamts8 and Serpinb5 were negatively correlated, while Ctgf, Etsl, PDGFRa, TGFP3and TIMP-2 were positively correlated. Overexpression of platelet-derived growth factor receptor alpha (PDGFRa) was found only in the endothelium of highly metastatic HCC, which is confirmed by RT-PCR, real time PCR and immunofluorescence.III. Preliminary study of PDGFRa as a predicative biomarker and therapeutic target for highly metastatic HCCImmunohistochemistry was employed to detect the distribution of PDGFRa in surgical specimens from 77 HCC patients having received curative resection. The results showed that PDGFRa expression closely associated with metastasis and recurrence of HCC. The positive rate of PDGFRa expression in recurrent cases was significantly higher than that in recurrent-free cases (31.1% vs. 9.4%; /?=0.028). Among 48 cases without microvessel invasion, PDGFRa-positive cases presented more frequently with recurrence when compared with PDGFRa-negative cases (80% vs. 36.8%; ^=0.029)In vitro and in vivo experiments were used to explore whether PDGFRa can be used as a therapeutic target for highly metastatic HCC. The treatment of STI571 (Gleevec), a protein tyrosine kinase inhibitor of PDGFR, ranging from 0.1 to 10 uM resulted in a mild increase in cytotoxicity of HCC tumor cells (MHCC97H) compared with control agent (p=0.062). The combined treatment of STI571 and interferon alpha (IFNa) did not increase the inhibition effect on cell proliferation compared with STI571 alone.Oral administration of STI571 inhibited the growth of highly metastatic HCC tumor by 27.3% (p=0.028) with a decrease of microvessel density by 35.1% (p=0.001). Combination therapy using STI571 and IFNa produced more effective reductions of tumor weight by 81.8% (p=0.000) and microvessel density by 70.2% (p=0.000).IV. The relationship between inhibition effect of IFNa on metastasis of HCC and tumor endotheliumIn vitro analyses of the effect of IFNa on TEC from highly metastatic HCC showed that the treatment of IFNa inhibited its function of low density lipoprotein internalization, but did not affect the movement of endothelial cells. IFNa (7.5 X 106 U/kg/d, s.c. injection) in vivo induced apoptosis of tumor endothelial cells in highly metastatic HCC tumor, and partly normalized the tumor blood vessel. Gene expression profiling of TEC from highly metastatic HCC, pretreated with IFNa in vivo, showed differential gene expression pattern. Several differentially expressed genes were identified, including PDGFRa, FGFR4, VEGF, IFN Y, Cox-2, TIMP-2 and TGF 3 3, etc. IFNa down-regulated the gene expression of not only some intrinsic growth factors but also some inhibitors of growth factors in TEC.Conclusions1. The modified immunomagnetic affinity purification can successfully isolate tumor endothelial cells from HCC.2. Differential gene expression does exist in endothelial cells derived from HCC with different metastatic potential.3. PDGFRa expression in tumor vessels cannot only be associated with metastatic potential in mouse model, but also in some human HCC with recurrence.4. Combination of STI571, PDGFR inhibitor, and IFNa can effectively inhibit the tumor burden of HCC in mice model.5. The inhibition effect of IFNa on HCC is associated with induction of apoptosis of tumor endothelial cells and normalization of tumor vessels. The underlying mechanism may be related to the changes of gene expression in tumor endothelial cells.The potential application of this work:1. The purified tumor endothelial cells can be widely used in the study of更多还原